This invention relates to methods for diagnosing vitamin B-12 deficiency. In particular, it is concerned with those methods for determining B-12 which include a step of separating the vitamin from substances present in body fluids which reversibly bind the vitamin.
Vitamin B-12 is transported in body fluids such as blood plasma by a group of .alpha. and .beta. globulins including transcobalamins I, II and III to which the vitamin is reversibly bound. These substances are hereinafter referred to as sample binding proteins. Presently favored vitamin B-12 assays treat the samples to release the vitamin from these binding proteins, usually by way of the concomitant irreversible denaturation of the proteins. The sample is then incubated with labelled vitamin B-12 and a limited amount of a specific vitamin B-12 binding protein such as intrinsic factor, whereby the labelled and sample vitamin compete for binding sites on the binding protein in proportion to their relative concentration. This specific binding protein is generally derived from a different source than the sample binding proteins; such specific binding proteins are well known and are characterized by high affinity and specificity for vitamin B-12. They are hereinafter referred to as reagent binding proteins. The mixture is then treated, to separate the protein bound and unbound vitamin, e.g. by adsorption of the free vitamin onto charcoal by immune or precipitation of the bound vitamin and followed by centrifugation. Either fraction is then assayed for the amount of label it contains.
The vitamin must be released from the sample binding proteins. If it is not, then that proportion of the vitamin which remains bound to the sample proteins will not be free to participate in the competition with labelled vitamin for the reagent binding protein. The result will be an incorrectly low value for the patient's total vitamin B-12. This competitive vitamin binding by test sample endogenous protein is henceforth termed sample binding. The amount of sample binding is determined by conducting the vitamin assay in the conventional fashion but in the absence of the reagent binding protein; any labelled vitamin which is located in the fraction in which the reagent binding protein would be normally found is a function of sample binding.
It is known to release a part of the bound vitamin by heating the test samples at 100.degree. C. to denature the sample binding proteins. However, Lee-Own et al. disclose that heat denaturing the samples excessively, i.e., heating at 100.degree. C. for more than about 5 minutes, will reinstate a portion of the binding activity. According to these authors protein reactivation can be prevented by adding dithiothreitol to the sample during the heating step.
We have found that heating in the presence of dithiothreitol fails to entirely eliminate sample binding in vitamin B-12 assays. For example, in our method we commonly encounter residual sample binding on the order of 20-25% when heat alone is used, and from 9-12% after heating with dithiothreitol.